Prenylation of cysteine sulfhydryls is a recently discovered post- translational modification. It is crucial to the membrane association and the biological activity of a series of signal transduction proteins, including ras proteins. Mutant ras proteins are involved in 20% of human cancers, and this fact has led to intense interest in prenylation inhibitors as potential cancer chemotherapeutic agents. In the initial phase of a program to study the nature of this critical modification pathway, we propose to use chemical means to address the following three questions. A) How specific are the binding interactions between farnesyl protein transferase (FPTase) and its prenyl donor, farnesyl pyrophosphate (FPP)? B) What is the mechanism employed by FPTase for the farnesylation of a cysteine residue of ras? C) How does the farnesyl cysteine moiety interact with its various membrane targets? It is anticipated that there will be a synergistic relationship among these three objectives, as synthetic efforts in one area will be directly applicable to another. The specific aims of this three year project are thus as follows: 1) Specifically substituted analogs of FPP will be prepared using our newly developed synthetic protocol (Tetrahedron Letters 1994, 35, 2509). They will then be assayed as substrates of or inhibitors for FPTase. 2) Analogs of FPP designed to be mechanism-based inhibitors will be prepared and their interaction with FPTase studied. If these substrates covalently modify FPTase, the labeled amino acid residues in the active site will then be identified. 3) Carbon-I 3 labeled farnesol and FPP will be synthesized and used to determine the conformation of these compounds in various solvents. A 13C- labeled farnesylated peptide will also be prepared to investigate the conformation of the farnesyl moiety in a membrane environment.